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Cellular and Molecular Life Sciences :... Aug 2022Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation,...
Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.
Topics: Animals; Histones; Male; Mammals; Meiosis; Metaphase; Mice; Phosphorylation; Semen; Spermatocytes
PubMed: 35930080
DOI: 10.1007/s00018-022-04507-8 -
Molecular Cell Sep 2020A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of...
A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.
Topics: Adenosine Triphosphatases; Anaphase; Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; DNA-Binding Proteins; Imaging, Three-Dimensional; Mammals; Metaphase; Mitosis; Prophase
PubMed: 32768407
DOI: 10.1016/j.molcel.2020.07.002 -
Cell Cycle (Georgetown, Tex.) Feb 2019PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and...
PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.
Topics: Animals; CDC2 Protein Kinase; Chromosomes; Cyclin B1; Cytoplasm; Female; M Phase Cell Cycle Checkpoints; Metaphase; Mice; Mice, Inbred ICR; Microinjections; Plasmids; Polar Bodies; Protein Kinase C beta; RNA Interference; RNA, Messenger; RNA, Small Interfering; Spindle Apparatus; Telophase
PubMed: 30730241
DOI: 10.1080/15384101.2018.1564492 -
ELife Jun 2021Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how...
Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin.
Topics: Anaphase; Animals; Bayes Theorem; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromosome Segregation; Chromosomes; Electron Microscope Tomography; Female; Katanin; Meiosis; Metaphase; Microtubules; Models, Theoretical; Oocytes; Spindle Apparatus
PubMed: 34114562
DOI: 10.7554/eLife.58903 -
Journal of Assisted Reproduction and... Sep 2019To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos.
PURPOSE
To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos.
METHODS
An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy.
RESULTS
Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3 ng/ml compared to < 1 ng/ml.
CONCLUSION
We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.
Topics: Adolescent; Adult; Amyloid; Animals; Blastocyst; Body Mass Index; Female; Humans; In Vitro Oocyte Maturation Techniques; Metaphase; Mice; Microscopy, Fluorescence; Middle Aged; Oocyte Retrieval; Oocytes; Ovulation Induction; Parthenogenesis; Pilot Projects; Prospective Studies; Young Adult; Zona Pellucida
PubMed: 31332596
DOI: 10.1007/s10815-019-01530-w -
Cell Cycle (Georgetown, Tex.) Feb 2021DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle... (Review)
Review
DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle stalls due to failed ATP hydrolysis, the onset of anaphase is delayed, presumably to allow extra time for decatenation to be completed. Recent evidence revealed that, unlike the spindle assembly checkpoint, this TopoII checkpoint response requires Aurora B and Haspin kinases and is triggered by SUMOylation of the C-terminal domain of TopoII.
Topics: Animals; Aurora Kinase B; Cell Cycle Proteins; DNA Topoisomerases, Type II; Genes, cdc; Humans; Intracellular Signaling Peptides and Proteins; M Phase Cell Cycle Checkpoints; Metaphase; Mitosis; Protein Serine-Threonine Kinases
PubMed: 33459116
DOI: 10.1080/15384101.2021.1875671 -
Cellular and Molecular Life Sciences :... Apr 2024Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule...
Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.
Topics: Animals; Mice; Biological Transport; Kinesins; Meiosis; Metaphase; Oocytes
PubMed: 38587639
DOI: 10.1007/s00018-024-05213-3 -
The Plant Cell Mar 2021The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are...
The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.
Topics: Arabidopsis; Kinesins; Kinetochores; Metaphase; Microtubules; Prophase
PubMed: 33751090
DOI: 10.1093/plcell/koaa003 -
Molecular Biology of the Cell Oct 2020Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator...
Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/C) form the main ubiquitin E3 ligase for these two proteins. APC/C is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation-defective CDC20 mutants. These CDC20 cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/C-dependent destruction of cyclin B in human cells.
Topics: Anaphase; Cdc20 Proteins; Chromosome Segregation; Cyclin B; HeLa Cells; Humans; Metaphase; Phosphorylation; Protein Processing, Post-Translational; Proteolysis; Receptors, Neuropeptide Y
PubMed: 32755477
DOI: 10.1091/mbc.E20-04-0252 -
Mutation Research. Genetic Toxicology... Jul 2021Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian...
Chromosomal aberrations (CAs) in peripheral blood lymphocytes can be used as biomarkers of cancer risk. Cytogenetic tests were conducted on 2396 healthy Hungarian individuals and cancer incidence was followed up from 1989 to 2018. Venous blood samples were obtained from the subjects and metaphases from lymphocyte cultures were prepared. We compared the CA frequencies of the various smoking (1-5; 6-10; 11-19; or 20-40 cigarettes/day) and exposure (irradiation; chemical industry; chemical research laboratory) groups. Chromatid break (p = 0.0002), total aberration (p = 0.002), and aberrant cell (p = 0.001) frequencies were higher in smokers than in non-smokers. For very heavy smokers, total CAs were significantly higher than for non-smokers (<0.001) or less intensive smokers (p = 0.003-0.0006). Intensity of smoking was a predictor of chromosomal aberrations, while duration was not. During follow-up, 177 (7.3 %) cancer cases were found. A Cox-regression model showed that subjects with cell values ≥2 CAs developed cancer more frequently (hazard ratio = 1.39; 95 % CI, 1.02-1.90). The relative risks of cancer were 1.06 (95 % CI 0.53-2.06) for light smokers and 1.74 (95 % CI 1.08-2.77) for very heavy smokers. The distributions of cancer sites showed differences between smoker and non-smoker groups: in male smokers, lung cancer, in non-smokers, prostate, and in females (both groups) breast cancer were most common. Cancer incidence correlated with chromosome aberrations; smoking was not a confounder in this relationship.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers; Cells, Cultured; Chromosome Aberrations; Female; Healthy Volunteers; Humans; Incidence; Lymphocytes; Male; Metaphase; Middle Aged; Neoplasms; Sister Chromatid Exchange; Smoking; Young Adult
PubMed: 34266629
DOI: 10.1016/j.mrgentox.2021.503373